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- PDB-7kyc: Structure of the S. cerevisiae phosphatidylcholine flippase Dnf1-... -

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Basic information

Entry
Database: PDB / ID: 7kyc
TitleStructure of the S. cerevisiae phosphatidylcholine flippase Dnf1-Lem3 complex in the E2P state
Components
  • Alkylphosphocholine resistance protein LEM3
  • Phospholipid-transporting ATPase DNF1
KeywordsTRANSLOCASE / P4 ATPase / Phosphatidylcholine Flippases
Function / homology
Function and homology information


regulation of vacuole organization / glycosylceramide flippase activity / mating projection tip membrane / aminophospholipid translocation / Ion transport by P-type ATPases / phosphatidylcholine flippase activity / phosphatidylserine flippase activity / ceramide translocation / ATPase-coupled intramembrane lipid transporter activity / phospholipid-translocating ATPase complex ...regulation of vacuole organization / glycosylceramide flippase activity / mating projection tip membrane / aminophospholipid translocation / Ion transport by P-type ATPases / phosphatidylcholine flippase activity / phosphatidylserine flippase activity / ceramide translocation / ATPase-coupled intramembrane lipid transporter activity / phospholipid-translocating ATPase complex / phosphatidylserine floppase activity / phosphatidylethanolamine flippase activity / cell septum / phosphatidylcholine floppase activity / cellular bud neck / P-type phospholipid transporter / phospholipid translocation / establishment or maintenance of cell polarity / Neutrophil degranulation / cell periphery / intracellular protein transport / endocytosis / cell surface receptor signaling pathway / endosome membrane / Golgi apparatus / magnesium ion binding / endoplasmic reticulum / ATP hydrolysis activity / mitochondrion / ATP binding / identical protein binding / plasma membrane / cytoplasm
Similarity search - Function
CDC50/LEM3 family / LEM3 (ligand-effect modulator 3) family / CDC50 family / P-type ATPase, subfamily IV / P-type ATPase, C-terminal / P-type ATPase, N-terminal / Phospholipid-translocating ATPase N-terminal / Phospholipid-translocating P-type ATPase C-terminal / Cation transport ATPase (P-type) / P-type ATPase, haloacid dehalogenase domain / P-type ATPase, phosphorylation site ...CDC50/LEM3 family / LEM3 (ligand-effect modulator 3) family / CDC50 family / P-type ATPase, subfamily IV / P-type ATPase, C-terminal / P-type ATPase, N-terminal / Phospholipid-translocating ATPase N-terminal / Phospholipid-translocating P-type ATPase C-terminal / Cation transport ATPase (P-type) / P-type ATPase, haloacid dehalogenase domain / P-type ATPase, phosphorylation site / P-type ATPase, cytoplasmic domain N / E1-E2 ATPases phosphorylation site. / P-type ATPase, A domain superfamily / P-type ATPase / P-type ATPase, transmembrane domain superfamily / HAD superfamily / HAD-like superfamily
Similarity search - Domain/homology
BERYLLIUM TRIFLUORIDE ION / Chem-POV / Phospholipid-transporting ATPase DNF1 / Phospholipid-transporting ATPase accessory subunit LEM3
Similarity search - Component
Biological speciesSaccharomyces cerevisiae (brewer's yeast)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.8 Å
AuthorsBai, L. / You, Q. / Jain, B.K. / Duan, H.D. / Kovach, A. / Graham, T.R. / Li, H.
Funding support United States, 1items
OrganizationGrant numberCountry
National Institutes of Health/National Cancer Institute (NIH/NCI)CA231466 to H.L. and GM107978 to T.R.G. United States
CitationJournal: Elife / Year: 2020
Title: Transport mechanism of P4 ATPase phosphatidylcholine flippases.
Authors: Lin Bai / Qinglong You / Bhawik K Jain / H Diessel Duan / Amanda Kovach / Todd R Graham / Huilin Li /
Abstract: The P4 ATPases use ATP hydrolysis to transport large lipid substrates across lipid bilayers. The structures of the endosome- and Golgi-localized phosphatidylserine flippases-such as the yeast Drs2 ...The P4 ATPases use ATP hydrolysis to transport large lipid substrates across lipid bilayers. The structures of the endosome- and Golgi-localized phosphatidylserine flippases-such as the yeast Drs2 and human ATP8A1-have recently been reported. However, a substrate-binding site on the cytosolic side has not been found, and the transport mechanisms of P4 ATPases with other substrates are unknown. Here, we report structures of the Dnf1-Lem3 and Dnf2-Lem3 complexes. We captured substrate phosphatidylcholine molecules on both the exoplasmic and cytosolic sides and found that they have similar structures. Unexpectedly, Lem3 contributes to substrate binding. The conformational transitions of these phosphatidylcholine transporters match those of the phosphatidylserine transporters, suggesting a conserved mechanism among P4 ATPases. Dnf1/Dnf2 have a unique P domain helix-turn-helix insertion that is important for function. Therefore, P4 ATPases may have retained an overall transport mechanism while evolving distinct features for different lipid substrates.
History
DepositionDec 7, 2020Deposition site: RCSB / Processing site: RCSB
Revision 1.0Jan 6, 2021Provider: repository / Type: Initial release
Revision 1.1Oct 30, 2024Group: Data collection / Database references / Structure summary
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / database_2 / em_admin / pdbx_entry_details / pdbx_modification_feature
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession ..._database_2.pdbx_DOI / _database_2.pdbx_database_accession / _em_admin.last_update / _pdbx_entry_details.has_protein_modification

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Assembly

Deposited unit
A: Phospholipid-transporting ATPase DNF1
B: Alkylphosphocholine resistance protein LEM3
hetero molecules


Theoretical massNumber of molelcules
Total (without water)229,73912
Polymers225,4912
Non-polymers4,24810
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

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Protein , 2 types, 2 molecules AB

#1: Protein Phospholipid-transporting ATPase DNF1 / Flippase DNF1


Mass: 178000.172 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Saccharomyces cerevisiae (strain ATCC 204508 / S288c) (yeast)
Strain: ATCC 204508 / S288c / Gene: DNF1, YER166W, SYGP-ORF7 / Production host: Saccharomyces cerevisiae S288C (yeast) / Strain (production host): ATCC 204508 / S288c
References: UniProt: P32660, P-type phospholipid transporter
#2: Protein Alkylphosphocholine resistance protein LEM3 / Brefeldin-A sensitivity protein 3 / Ro-sensitive 3


Mass: 47490.395 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Saccharomyces cerevisiae (strain ATCC 204508 / S288c) (yeast)
Strain: ATCC 204508 / S288c / Gene: LEM3, BRE3, ROS3, YNL323W, N0333 / Production host: Saccharomyces cerevisiae S288C (yeast) / Strain (production host): ATCC 204508 / S288c / References: UniProt: P42838

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Sugars , 3 types, 5 molecules

#3: Polysaccharide alpha-D-mannopyranose-(1-4)-2-acetamido-2-deoxy-beta-D-glucopyranose-(1-4)-2-acetamido-2-deoxy-beta- ...alpha-D-mannopyranose-(1-4)-2-acetamido-2-deoxy-beta-D-glucopyranose-(1-4)-2-acetamido-2-deoxy-beta-D-glucopyranose


Type: oligosaccharide / Mass: 586.542 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
DescriptorTypeProgram
DManpa1-4DGlcpNAcb1-4DGlcpNAcb1-ROHGlycam Condensed SequenceGMML 1.0
WURCS=2.0/2,3,2/[a2122h-1b_1-5_2*NCC/3=O][a1122h-1a_1-5]/1-1-2/a4-b1_b4-c1WURCSPDB2Glycan 1.1.0
[][D-1-deoxy-GlcpNAc]{[(4+1)][b-D-GlcpNAc]{[(4+1)][a-D-Manp]{}}}LINUCSPDB-CARE
#4: Polysaccharide 2-acetamido-2-deoxy-beta-D-glucopyranose-(1-4)-2-acetamido-2-deoxy-beta-D-glucopyranose


Type: oligosaccharide / Mass: 424.401 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
DescriptorTypeProgram
DGlcpNAcb1-4DGlcpNAcb1-ROHGlycam Condensed SequenceGMML 1.0
WURCS=2.0/1,2,1/[a2122h-1b_1-5_2*NCC/3=O]/1-1/a4-b1WURCSPDB2Glycan 1.1.0
[][D-1-deoxy-GlcpNAc]{[(4+1)][b-D-GlcpNAc]{}}LINUCSPDB-CARE
#8: Sugar ChemComp-NAG / 2-acetamido-2-deoxy-beta-D-glucopyranose / N-acetyl-beta-D-glucosamine / 2-acetamido-2-deoxy-beta-D-glucose / 2-acetamido-2-deoxy-D-glucose / 2-acetamido-2-deoxy-glucose / N-ACETYL-D-GLUCOSAMINE


Type: D-saccharide, beta linking / Mass: 221.208 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C8H15NO6
IdentifierTypeProgram
DGlcpNAcbCONDENSED IUPAC CARBOHYDRATE SYMBOLGMML 1.0
N-acetyl-b-D-glucopyranosamineCOMMON NAMEGMML 1.0
b-D-GlcpNAcIUPAC CARBOHYDRATE SYMBOLPDB-CARE 1.0
GlcNAcSNFG CARBOHYDRATE SYMBOLGMML 1.0

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Non-polymers , 3 types, 5 molecules

#5: Chemical ChemComp-BEF / BERYLLIUM TRIFLUORIDE ION


Mass: 66.007 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: BeF3
#6: Chemical ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: Mg
#7: Chemical ChemComp-POV / (2S)-3-(hexadecanoyloxy)-2-[(9Z)-octadec-9-enoyloxy]propyl 2-(trimethylammonio)ethyl phosphate / POPC


Mass: 760.076 Da / Num. of mol.: 3 / Source method: obtained synthetically / Formula: C42H82NO8P / Comment: phospholipid*YM

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Details

Has ligand of interestN
Has protein modificationY

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction

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Sample preparation

ComponentName: S. cerevisiae phosphatidylcholine flippase Dnf1-Lem3 complex in the E2P state
Type: COMPLEX / Entity ID: #1-#2 / Source: RECOMBINANT
Source (natural)Organism: Saccharomyces cerevisiae (strain ATCC 204508 / S288c) (yeast)
Strain: ATCC 204508 / S288c
Source (recombinant)Organism: Saccharomyces cerevisiae (strain ATCC 204508 / S288c) (yeast)
Strain: ATCC 204508 / S288c
Buffer solutionpH: 7.4
SpecimenConc.: 1.5 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
VitrificationCryogen name: ETHANE

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: OTHER
Electron lensMode: BRIGHT FIELD
Image recordingElectron dose: 64 e/Å2 / Film or detector model: GATAN K3 (6k x 4k)

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Processing

CTF correctionType: NONE
3D reconstructionResolution: 2.8 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 590043 / Symmetry type: POINT

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